International Journal of Food Microbiology 153 (2012) 66–72

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Antifungal activity by vapor contact of essential oils added to amaranth, chitosan, or

starch edible films
Raúl Avila-Sosa a, b, Enrique Palou a, María Teresa Jiménez Munguía a,
Guadalupe Virginia Nevárez-Moorillón c, Addí Rhode Navarro Cruz b, Aurelio López-Malo a,⁎
a
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Cholula, Pue. 72810, México
b
Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, 14 Sur y Av. San Claudio, Ciudad Universitaria, Puebla, Pue. 72420, México
c
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, P.O. Box 1542-C Chihuahua, Chih., México

a r t i c l e i n f o a b s t r a c t

Article history: Antimicrobial agents can be incorporated into edible films to provide microbiological stability, since films can be
Received 13 April 2011 used as carriers of a variety of additives to extend product shelf life and reduce the risk of microbial growth on
Received in revised form 3 October 2011 food surfaces. Addition of antimicrobial agents to edible films offers advantages such as the use of small antimi-
Accepted 22 October 2011
crobial concentrations and low diffusion rates. The aim of this study was to evaluate inhibition by vapor contact
Available online 30 October 2011
of Aspergillus niger and Penicillium digitatum by selected concentrations of Mexican oregano (Lippia berlandieri
Keywords:
Schauer), cinnamon (Cinnamomum verum) or lemongrass (Cymbopogon citratus) essential oils (EOs) added to
Natural antimicrobials amaranth, chitosan, or starch edible films. Essential oils were characterized by gas chromatography–mass spec-
Edible films trometry (GC/MS) analysis. Amaranth, chitosan and starch edible films were formulated with essential oil con-
Vapor contact centrations of 0.00, 0.25, 0.50, 0.75, 1.00, 2.00, or 4.00%. Antifungal activity was evaluated by determining the
Essential oils mold radial growth on agar media inoculated with A. niger and P. digitatum after exposure to vapors arising
from essential oils added to amaranth, chitosan or starch films using the inverted lid technique. The modified
Gompertz model adequately described mold growth curves (mean coefficient of determination 0.991 ± 0.05).
Chitosan films exhibited better antifungal effectiveness (inhibition of A. niger with 0.25% of Mexican oregano
and cinnamon EO; inhibition of P. digitatum with 0.50% EOs) than amaranth films (2.00 and 4.00% of cinnamon
and Mexican oregano EO were needed to inhibit the studied molds, respectively). For chitosan and amaranth
films a significant increase (pb 0.05) of lag phase was observed among film concentrations while a significant
decrease (pb 0.05) of maximum specific growth was determined. Chitosan edible films incorporating Mexican
oregano or cinnamon essential oil could improve the quality of foods by the action of the volatile compounds
on surface growth of molds.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction mixtures of several components. The inherent aroma and antimicro-

bial activity of EOs are related commonly to the chemical structure
Application of antimicrobial agents could prevent or delay micro- of their components, the concentration in which the components
bial spoilage of food products; nevertheless advances in biological are present, and the interactions among them affecting their bioactive
and chemical analytical methods have raised questions regarding properties. Some studies have demonstrated that EOs have greater
the safety of some chemical food additives (Avila-Sosa et al., 2010a). antibacterial activity than their major constituents when tested sepa-
Because of concerns related to the use of chemical additives, con- rately (Burt, 2004). Since EOs are generally recognized as safe (GRAS)
sumers prefer natural additives (including antimicrobial agents) for use as flavor additives (Tunc et al., 2007), the possibility of utiliz-
incorporated into food products (Davidson, 2001). ing their antimicrobial effects by adding EOs to the surface of food is
Several research groups are studying the chemical structure being investigated (Ayala-Zavala et al., 2009; Brul and Coote, 1999;
and activity of natural antimicrobials from fruits, vegetables, grains, Holley and Patel, 2005). Despite the high efficacy of the EOs and
herbs, and spices. Efforts have concentrated on the use of natural their constituents against food-borne pathogens and spoilage micro-
extracts, such as those obtained from spices. Essential oils (EOs) are organisms when in vitro tests are conducted, the same effect in food
aromatic oily liquids obtained from plant material and are usually products is only achieved when higher concentrations of EOs are uti-
lized. When EOs are applied directly on a food surface by dipping,
powdering or spraying, their highly hydrophobic and volatile active
⁎ Corresponding author. Tel.: + 52 222 229 2126; fax: + 52 222 229 2727. substances are bound by food components, while other components
E-mail address: [email protected] (A. López-Malo). of the EOs are partitioned through the product according to their

0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2011.10.017
 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72 67

affinity with water. To avoid this problem an alternative is the incor- 220 °C, and held at the final temperature for 10 min, for a total run
poration of essential oil within edible films. Edible films can serve as time of 30 min. The obtained spectra were compared with respective
carriers releasing antimicrobials onto the food surface; controlling mass spectra of pure compounds, and also with the mass profile of
microbial growth. Other advantages of edible films are the ability to the same compounds available from the US National Institute of
delay transmission of moisture, oxygen, aroma and solutes, thus Standard Technology (NIST) library (Avila-Sosa et al., 2010b).
enhancing product shelf life (Cha and Chinnan, 2004; Goni et al.,
2009; Guilbert et al., 2002; Krochta, 1997; Marsh and Bugusu, 2007). 2.2. Edible film preparation
Edible films can reduce antimicrobial diffusion into the product
since the essential oil forms part of the chemical structure of the Studied edible films were made by the casting method, which con-
film and interacts with the polymer and the plasticizer. Antimicrobial sists of drying the corresponding film forming solution (FFS) that had
release from the edible film depends on many factors, including elec- been applied on a support. FFSs were formulated with amaranth flour
trostatic interactions between the antimicrobial agent and the poly- (bought at a local market in Puebla, Mexico) with water solution of
mer chains, osmosis, structural changes induced by the presence of 4% w/w amaranth flour, pH was adjusted to 10.7 with NaOH (0.1 N)
antimicrobial, and environmental conditions. Compared with direct (Tapia-Blácido et al., 2005). Medium molecular weight (450 kDa) of
application, smaller amounts of antimicrobial agents would be needed chitosan (Aldrich Chemical Co. Milwaukee, WI) was prepared with
when edible films are used in order to achieve a specific shelf life 1.5% w/w chitosan in 1.5% v/v acetic acid, the solution was stirred
due to a gradual release on food surfaces (Ponce et al., 2008; Sebti overnight at room temperature. High amylose corn starch (CPI Ingre-
et al., 2005). dients, Mexico) was used with 1 g of starch, in previously sterilized
Many reported data focus on the discovery of new natural antimi- 10 ml 0.25 N sodium hydroxide and 10 ml distilled water. FFS were
crobials that can be incorporated into edible films. A number of com- maintained 60 min under stirring conditions (Bertuzzi et al., 2007).
pounds have been proposed to have antimicrobial activity in foods, Amaranth and chitosan solutions were sterilized at 121 °C for
including organic acids, enzymes, bacteriocins, and essential oils 15 min. To enable film formation, glycerol (Aldrich Chemical Co.
(Cagri et al., 2004). In a previous paper (Avila-Sosa et al., 2010b) we Milwaukee, WI) 1.3% v/v and Tween 20 (Aldrich Chemical Co.
evaluated by direct contact the antifungal effect of Mexican oregano Milwaukee, WI) 0.5% v/v were added as plasticizers for amaranth
essential oil incorporated into different edible films providing evidence and chitosan films respectively. Starch FFS was gelatinized in a shaker
of inhibition of spoilage microorganisms (A. niger and Penicillium spp.). water bath at 78–80 °C for 10 min; when the solution was near 40 °C,
Some studies have been published regarding inhibition of several glycerol (1.2% v/v) was added (Zivanovic et al., 2005).
microorganisms by the vapor-phase of EOs (Edris, 2007; Inouye et al., FFSs were mixed (IKA High Performance Disperser T18, Chicago,
2000, 2001, 2006; Inouye, 2003; López et al., 2005, 2007; Nielsen and IL) under aseptic conditions at 20,000 rpm for 1 min at room temper-
Rios, 2000; Suppakul et al., 2003). Until now, no standard assay exists ature with the incorporation of EOs at 0.00%, 0.25%, 0.50%, 0.75%,
to evaluate microbial inhibition by vapor contact of EOs. There are 1.00%, 2.00%, or 4.00% (v/v) final concentration and poured into
many methods reported by different authors (Kloucek et al., in press) 60 mm inner diameter sterile Petri dishes covers. Films were prepared
the most common are: 1) the overlay method (Du et al., 2008, 2009b) with 7 ml of FFS per Petri dish (1 film), dried under 0.35 kg/cm 2
which is just a simple modification of the disk diffusion assay and can vacuum at 30 °C for 12 h. Films were kept in sealed Petri dishes at
be related to direct contact of the films on food surfaces and 2) the 4 °C until analysis (Avila-Sosa et al., 2010b).
vapor phase method (López et al., 2007) where EOs and microorgan-
isms are placed separately in some sealed environment and can be 2.3. Thickness
related to microbial inhibition from a distance without direct contact
of the film with the contaminated food. Film thickness (μm) was determined on every film formulation,
The aim of this study was to evaluate inhibition of A. niger and reporting means of measurements at 5 points of every film using a
P. digitatum by vapor contact with selected concentrations of Mexican micrometer (Scala, 111-B, Mexico City, Mexico).
oregano, cinnamon or lemongrass EOs added to amaranth, chitosan, or
starch edible films. 2.4. Fungal strains and spore suspensions

2. Materials and methods A. niger and P. digitatum were obtained from Universidad de las
Américas Puebla Food Microbiology Laboratory. They were cultivated
2.1. Essential oils and chemical characterization for 5 days at 25 °C on potato-dextrose agar plates (PDA, Merck,
Mexico) acidified with 10% tartaric acid, then were used to recover
Mexican oregano (L. berlandieri Schauer) essential oil was pro- fungal spores by pouring 9 ml of sterile physiological water (0.90% w/v
vided by CiReNA (Natural Resources Research Center of Salaices, of NaCl) on the agar plate surface, followed by a gentle scraping
López, Chihuahua, Mexico). Cinnamon bark (Cinnamomum verum) using a sterile rake to remove the maximum quantity of spores. Spore
and lemongrass (Cymbopogon citratus) were bought at a local market suspensions were transferred into sterile tubes. The number of spores
in Puebla, Mexico. Plant material was authenticated by botanists of present in the suspension was determined using a hemocytometer and
the Department of Biology of Benemerita Universidad Autonoma de an optical microscope (Zeiss Primo Star, Göttingen, Germany), and
Puebla, Puebla, Mexico by visual inspection and comparison with expressed as number of spores per milliliter (spores/ml). Suspensions
the herbarium reference by data base. Lemongrass was washed and were serially diluted to approximately 1×106 spores/ml (Sebti et al.,
air-dried at room temperature for 3 days and then finely ground 2005).
with a mortar and pestle. All EOs were obtained by vapor distillation
for 4 h with a Cleavenger-type apparatus. EOs were analyzed with 2.5. Vapor contact assay
a GC Perkin Elmer Turbo Mass Gold MS-Auto system XL™ (Perkin-
Elmer, Norwalk, CT) with a splitless injector and an FID detector, The inverted lid technique was used (Du et al., 2009a); 10 μl of
equipped with an AT-1 capillary column (30 m ∗ 0.25 I.D. ∗ 0.25 μm). spore suspension (1 × 10 6 spores/ml) were placed in the center of
Helium was used as the carrier gas, and the following conditions the PDA plate and were dried in a laminar flow hood under aseptic
were set for the analysis: injector, 100 °C, detector 225 °C; the initial conditions (Labconco, Kansas City, MO, USA) at room temperature
oven temperature was 55 °C held for 1 min, followed by a ramp-up for 30 min, then Petri dish cover with the corresponding antimicro-
of 3 °C/min up to 95 °C, and a second ramp-up of 25 °C/min up to bial film was placed to cover the plate with inoculated agar. Radial
68 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72

growth was measured every 24 h during 8 days of incubation 25 °C. A while others were detected in very low concentrations, such as
growth control was prepared in parallel, in order to ensure that viable p-cymene, 1, 8-cineole, and γ-terpinen. Cinnamon EO chemical compo-
organisms were present. Every test was performed in triplicate. sition presents eugenol (3.402 g/ml), cinnamaldehyde (0.652 g/ml)
and acetoeugenol as major constituents. Finally lemongrass GC-MS
2.6. Fungal growth modeling and statistical analysis exhibited geranial (2.873 g/ml) and geranial acetate as major compo-
nents of this essential oil.
Growth data were fitted using the modified Gompertz model as Figs. 1, 2, and 3 present the change in mold colony diameter under
reported by Char et al. (2007): the effect of amaranth, chitosan, and starch edible films respectively,
  with added Mexican oregano, cinnamon or lemongrass EOs. The mod-
D ified Gompertz model adequately fitted the experimental data (mean
Ln t ¼ A expf− exp½ðυm ⋅e=AÞðλ−t Þ þ 1g ð1Þ
Do coefficient of determination 0.991 ± 0.05). Gompertz model can be
utilized to describe mold radial growth, despite the fact that this
where: Dt (cm) is the average colony diameter at time t (day), and model was originally proposed for bacterial growth (Char et al.,
Do (cm) is the average colony diameter at initial time; A is the maxi- 2007). Calculated growth parameters were useful to compare anti-
mum mold growth achieved during the stationary phase, υm is the fungal effects among concentrations of the studied EOs in the edible
maximum specific growth rate (1/day), λ is the lag phase (day) and films tested.
e = exp (1). For A. niger, amaranth edible films exhibited the lowest antifungal
Statistical analyses were performed with the General Linear Model effectiveness since only high concentrations of cinnamon (2.00%) and
procedure in Minitab 15 (LEAD Technologies Inc., NJ). Data were Mexican oregano (4.00%) EOs exerted antifungal effects (Fig. 1A and
obtained by triplicate with each replicate value obtained from an in- Table 1). Gompertz parameters (Table 1) for amaranth edible films
dividual experiment. Significantly (p b 0.05) different means were with cinnamon (1.00%) and Mexican oregano EOs (2.00%) showed
separated with Tukey's test. significant differences (p b 0.05) in lag phase values with respect to
amaranth films formulated without EO. In the case of P. digitatum,
3. Results amaranth films (Fig. 1B) exhibited growth inhibition at cinnamon
EO concentrations of 2.00%; for Mexican oregano and lemongrass
Chemical analysis of Mexican oregano EO distinguished several com- EOs at higher concentrations, higher maximum specific growth
pounds, particularly thymol (2.103 g/ml) and carvacrol (0.533 g/ml), rates were observed which were statistically different (p b 0.05)

A 2.5 A 2.5
2 2
Ln(Dt/Do)

Ln(Dt/Do)

1.5 1.5

1 1

0.5 0.5

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (Days) Time (Days)

B 2.5 B 2.5
2 2
Log(Dt/Do)

Ln(Dt/Do)

1.5 1.5

1 1

0.5 0.5

0 0
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time (Days) Time (Days)
Fig. 1. Effect of amaranth edible films added with essential oils at selected concentra-
tions [0.00% (▲), cinnamon 1.00% (■), Mexican oregano 2.00% (●), Mexican oregano Fig. 2. Effect of chitosan edible films added with essential oils at selected concentra-
4.00% (Δ), or lemongrass 4.00% (□)] on Aspergillus niger (A) and Penicillium digitatum tions [0.00% (▲), cinnamon 0.25% (■), Mexican oregano 0.25% (●), or lemongrass
(B) growth. Dt is the average colony diameter at time t and Do is the average colony di- 4.00% (□)] on Aspergillus niger (A) and Penicillium digitatum (B) growth. Dt is the aver-
ameter at initial time. age colony diameter at time t and Do is the average colony diameter at initial time.
 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72 69

A from films without EO (Table 2). The thickness of amaranth films

2.5 (14.333 ± 1.479 μm) was not significantly different (p b 0.05) when
the concentration of the EOs increased.
2 Chitosan films exhibited the greatest inhibition against A. niger
and P. digitatum. Inhibition was achieved at low concentrations of
cinnamon and Mexican oregano EOs, statistical differences (p b 0.05)
Log(Dt/Do)

1.5 were observed among υm and λ (Tables 1 and 2) parameters. Lemon-

grass EO exhibited no inhibition effect against A. niger, however a sig-
1 nificant increase (p b 0.05) in the lag phase (Fig. 2A, Table 2) was
observed when EO concentration increased. Chitosan film thickness
varied from 5.33 ± 0.836 to 13.26 ± 0.414 μm.
0.5
A. niger inhibition was obtained with starch edible films with con-
centrations of 0.50% cinnamon, 2.00% Mexican oregano and 4.00%
0 lemongrass EOs (Table 1). Significant differences (p b 0.05) in the lag
0 1 2 3 4 5 6 7 8 phase (Fig. 3A) among 0.00%, 0.25% of cinnamon, 1.00% of Mexican
Time (Days) oregano, and 0.50% of lemongrass concentrations were observed
(Table 1). A similar effect on P. digitatum growth is seen in Fig. 3B.
B 2.5 Films formulated with Mexican oregano presented inhibition at a
concentration of 0.75%. For starch films when the concentration of
essential oil increased, the film thickness varied from 11.533 ± 0.547
2 to 22.066 ± 0.435 μm.
Ln(Dt/Do)

1.5 4. Discussion

The present study has demonstrated the potential of EOs added

1
to edible films as antifungal agents by vapor contact. Antimicrobial
activities of the different EO vapors could be achieved at lesser amounts
0.5 than when applying the EOs in direct contact with a food surface.
Mexican oregano and cinnamon EO were the most effective, their activ-
0 ity due to their constituents as revealed during chemical characteriza-
0 1 2 3 4 5 6 7 8 tion. Determined concentrations of components of Mexican oregano
Time (Days) EO concur with previous reports (Avila-Sosa et al., 2010b; Dunford
and Silva-Vazquez, 2005; Silva-Vazquez and Dunford, 2005). The main
Fig. 3. Effect of starch edible films added with essential oils at selected concentrations component of Mexican oregano EO is thymol, and its concentration is
[0.00% (▲), cinnamon 0.25% (■), Mexican oregano 0.50% (●), Mexican oregano 1.00% greater when the extracts are obtained from younger plants (Arcila-
(Δ), or lemongrass 2.00% (□)] on Aspergillus niger (A) and Penicillium digitatum (B) Lozano et al., 2004; Avila-Sosa et al., 2010a). In contrast, in essential
growth. Dt is the average colony diameter at time t and Do is the average colony diam-
oils from European oregano the concentration of carvacrol is much
eter at initial time.
higher (70–88%) than that of thymol (2%) (Aligiannis et al., 2001;

Table 1
Modified Gompertz model parameters+ (mean ± standard deviation) for Aspergillus niger growth curves subjected to selected concentrations of Mexican oregano (Lippia berlandieri
Schauer), cinnamon (Cinnamomum verum), or lemongrass (Cymbopogon citratus) essential oils added to amaranth, chitosan, or starch edible films by vapor contact.

Edible film Gompertz parameters

A υm (1/day) λ (day) A υm (1/day) λ (day) A υm (1/day) λ (day)

Amaranth Chitosan Starch

Without essential oil 2.21a ± 0.04 2.46a ± 0.09 0.15a ± 0.01 2.40a ± 0.02 0.85a ± 0.01 0.00a ± 0.01 2.21a ± 0.01 1.59a ± 0.09 0.07a ± 0.03
Cinnamon 0.25% 2.34a ± 0.12 2.52a ± 0.13 0.23a ± 0.05 –⁎ –⁎ > 7.00b,⁎ 1.34b ± 0.18 1.07b ± 0.04 2.12b ± 0.13
Cinnamon 0.50% 2.41a ± 0.18 2.66a ± 0.16 0.31a ± 0.08 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 0.75% 2.38a ± 0.09 2.61a ± 0.11 0.27a ± 0.02 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 1.00% 1.99b ± 0.12 2.30a ± 0.16 2.14b ± 0.02 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 2.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 4.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 0.25% 2.37a ± 0.09 3.16a ± 0.05 0.52a ± 0.01 –⁎ –⁎ > 7.00b,⁎ 1.94a ± 0.03 1.45a ± 0.12 0.12a ± 0.02
Oregano 0.50% 2.28a ± 0.11 2.96a ± 0.08 0.45a ± 0.13 –⁎ –⁎ > 7.00b,⁎ 1.98a ± 0.16 1.32a ± 0.16 0.27a ± 0.08
Oregano 0.75% 2.98a ± 0.07 2.99a ± 0.23 0.33a ± 0.11 –⁎ –⁎ > 7.00b,⁎ 2.04a ± 0.11 1.47a ± 0.01 0.33a ± 0.08
Oregano 1.00% 2.45a ± 0.22 3.10a ± 0.16 0.39a ± 0.08 –⁎ –⁎ > 7.00b,⁎ 1.91a ± 0.20 1.96c ± 0.24 1.13d ± 0.04
Oregano 2.00% 1.96b ± 0.18 1.96b ± 0.25 1.15d ± 0.07 –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 4.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ > 7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Lemongrass 0.25% 2.13a ± 0.04 2.63a ± 0.09 0.40a ± 0.01 2.40a ± 0.02 0.85a ± 0.01 0.00a ± 0.01 1.78a ± 0.21 1.33a ± 0.15 0.17a ± 0.01
Lemongrass 0.50% 2.03a ± 0.02 2.51a ± 0.15 0.44a ± 0.08 2.40a ± 0.03 0.85a ± 0.02 0.00a ± 0.02 1.93a ± 0.14 1.41a ± 0.12 0.22a ± 0.05
Lemongrass 0.75% 2.17a ± 0.07 2.55a ± 0.18 0.41a ± 0.04 2.40a ± 0.04 0.85a ± 0.03 0.00a ± 0.03 2.01a ± 0.11 1.55a ± 0.18 0.21a ± 0.08
Lemongrass 1.00% 2.07a ± 0.01 2.62a ± 0.21 0.38a ± 0.08 2.40a ± 0.05 0.85a ± 0.04 0.00a ± 0.04 2.09a ± 0.07 1.64a ± 0.11 0.19a ± 0.01
Lemongrass 2.00% 2.31a ± 0.18 2.48a ± 0.13 0.23a ± 0.02 2.40a ± 0.06 0.85a ± 0.05 0.00a ± 0.05 2.13a ± 0.13 2.40d ± 0.28 1.14d ± 0.02
Lemongrass 4.00% 1.94b ± 0.05 1.84b ± 0.34 0.12a ± 0.05 2.29a ± 0.02 2.65b ± 0.24 2.16c ± 0.01 – –⁎ >7.00c,⁎

Means followed by a different superscript letter within a column for each studied film (amaranth, chitosan or starch) are significantly different (p b 0.05).
+
A: maximum mold growth in the stationary phase; υm: maximum specific growth rate; λ: lag phase.
⁎ No growth.
70 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72

Table 2
Modified Gompertz model parameters+ (mean ± standard deviation) for Penicillium digitatum growth curves subjected to selected concentrations of Mexican oregano (Lippia
berlandieri Schauer), cinnamon (Cinnamomum verum), or lemongrass (Cymbopogon citratus) essential oils added to amaranth, chitosan, or starch edible films by vapor contact.

Edible film Gompertz parameters

A υm (1/day) λ (day) A υm (1/day) λ (day) A υm (1/day) λ (day)

Amaranth Chitosan Starch

Without essential oil 1.97a ± 0.08 1.10a ± 0.45 0.13a ± 0.03 2.14a ± 0.01 1.91a ± 0.01 0.09a ± 0.01 1.90a ± 0.12 2.42b ± 0.16 0.09a ± 0.02
Cinnamon 0.25% 1.90a ± 0.02 0.96a ± 0.12 0.22a ± 0.07 1.62b ± 0.08 1.46b ± 0.04 0.10a ± 0.05 1.15b ± 0.21 1.67a ± 0.07 2.48b ± 0.03
Cinnamon 0.50% 1.89a ± 0.07 1.05a ± 0.36 0.45a ± 0.18 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 0.75% 1.95a ± 0.04 1.23a ± 0.09 0.34a ± 0.09 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 1.00% 1.82b ± 0.02 3.17b ± 0.29 2.17b ± 0.01 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 2.00% –⁎ –⁎ >7.00c,⁎ –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Cinnamon 4.00% –⁎ –⁎ >7.00c,⁎ 1.81c ± 0.05 0.98b ± 0.07 0.05a ± 0.05 –⁎ –⁎ >7.00c,⁎
Oregano 0.25% 2.02a ± 0.02 1.32a ± 0.35 0.42a ± 0.06 –⁎ –⁎ >7.00b,⁎ 0.96b ± 0.15 0.98c ± 0.19 2.35b ± 0.05
Oregano 0.50% 1.94a ± 0.09 0.93a ± 0.04 0.54a ± 0.11 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 0.75% 1.97b ± 0.10 1.22a ± 0.17 0.33a ± 0.05 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 1.00% 2.10b ± 0.05 1.17a ± 0.12 0.14 ± 0.02 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 2.00% 2.03b ± 0.01 1.30a ± 0.09 0.49a ± 0.12 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Oregano 4.00% 1.71c ± 0.09 2.23c ± 0.23 1.85b ± 0.58 –⁎ –⁎ >7.00b,⁎ –⁎ –⁎ >7.00c,⁎
Lemongrass 0.25% 1.95b ± 0.03 1.23b ± 0.08 0.43a ± 0.12 2.09a ± 0.01 1.99a ± 0.05 0.11a ± 0.08 1.99a ± 0.10 2.53b ± 0.16 0.14a ± 0.11
Lemongrass 0.50% 1.99b ± 0.09 1.37b ± 0.16 0.39a ± 0.07 1.97a ± 0.20 1.83a ± 0.11 0.08a ± 0.04 1.88a ± 0.01 2.61b ± 0.06 0.08a ± 0.01
Lemongrass 0.75% 2.10b ± 0.16 1.06b ± 0.22 0.47a ± 0.11 2.04a ± 0.03 1.96a ± 0.08 0.12a ± 0.03 1.93a ± 0.04 2.32b ± 0.22 0.12a ± 0.02
Lemongrass 1.00% 1.99b ± 0.05 1.29b ± 0.42 0.29a ± 0.16 1.88a ± 0.11 1.97a ± 0.12 0.10a ± 0.06 1.97a ± 0.13 2.39b ± 0.14 0.14a ± 0.04
Lemongrass 2.00% 1.92b ± 0.14 1.33b ± 0.26 0.33a ± 0.19 2.44a ± 0.15 1.91a ± 0.03 0.09a ± 0.02 1.74c ± 0.08 2.48b ± 0.05 1.13d ± 0.03
Lemongrass 4.00% 1.76c ± 0.07 1.91d ± 0.22 1.17d ± 0.01 2.21a ± 0.06 3.20c ± 0.32 0.06a ± 0.05 –⁎ –⁎ >7.00c,⁎

Means followed by a different superscript letter within a column for each studied film (amaranth, chitosan or starch) are significantly different (p b 0.05).
+
A: maximum mold growth in the stationary phase; υm: maximum specific growth rate; λ: lag phase.
⁎ No growth.

Chorianopoulos and Kalpoutzakis, 2004). The antimicrobial activity of A. niger with cinnamon EO; fungal sporulation was completely
oregano EO is due to the presence of both thymol and carvacrol along retarded at concentrations of 0.50%. However, at lower concentra-
with other minor components (Lambert et al., 2001; Sivropoulou et tions spore germination was accelerated.
al., 1996). Inouye et al. (2001), reported concentrations of cinnamalde- Some investigators incorporated EOs from herbs and spices (such
hyde (63%) in cinnamon bark oil as main component, however the oil as cinnamon and lemongrass) in active packaging polypropylene
from leaves and bark of some cinnamon bushes has eugenol as the films on bakery products, inhibiting by vapor contact Penicillium
main component (Ayala-Zavala et al., 2009). Chericoni et al. (2005) commune and A. niger at 2.00% of cinnamon EO, thus increasing the
reported a similar chemical composition for cinnamon EO as that product shelf life more than three times (Gutiérrez et al., 2009).
obtained in our study. The main component of lemongrass EO is Recently, Du et al. (2009a, 2009b) incorporated allspice, garlic and
geranial with similar concentrations as previously reported (Inouye, oregano EOs in tomato films and demonstrated the inhibition by
2003; Masuda et al., 2008; Schaneberg and Khan, 2002). Although vapor phase of E. coli O157:H7, Salmonella enterica, and Listeria mono-
major active compounds are known to be responsible for the antimicro- cytogenes. We studied the inhibition of A. niger and Penicillium sp. by
bial activity displayed by EOs, some studies reported that minor direct contact of amaranth, chitosan, and starch edible films added
compounds might also have synergistic or additive effects (Tyagi and with Mexican oregano EO; finding inhibition at 0.50% on starch and
Malik, 2011). chitosan films (Avila-Sosa et al., 2010b).
We have not found any other reports of fungal inhibition by vapor The degree of fungal inhibition by vapor contact of different EOs
contact of EOs incorporated into edible films, however there are many incorporated into the three studied edible films depended directly
studies that demonstrated that EOs have antifungal activity at higher on the type of polymer used to form the film, as different polymers
concentrations than those determined here. Inouye et al. (2001, retain EOs to different degrees. Amaranth films were least effective
2006) and Inouye (2003) evaluated the inhibition of a variety of as carriers for the tested EOs. Amaranth contains starch (62%) and
molds including Aspergillus fumigatus, and A. niger by cinnamon and proteins (14%); a combination of these components is ideal for the
lemongrass EOs and mentioned that EOs affected the three stages of formation of edible films (Tapia-Blácido et al., 2005, 2007). Given
the life cycle of filamentous fungi. A fungicidal effect of cinnamon the presence of different kinds of components in the edible films,
and European oregano EOs in Penicillium islandicum and Aspergillus the main compounds of the tested EOs (thymol, carvacrol, eugenol
flavus with concentrations near 0.50% has been reported (López et and geranial) could be interacting with these components, potentially
al., 2005, 2007). Mixtures of cinnamon and clove EOs against A. flavus affecting the antimicrobial activity, thus no significant differences
were tested at ranges of 1.00–2.00% and results indicated that the (p > 0.05) were observed in maximum growth rate and lag phase
antifungal activity of EOs depends on the experimental assay utilized values at low concentrations of EOs. In order to counteract this effect,
(López et al., 2005, 2007). The inhibitory effects of EOs were greater higher concentrations of EOs must be added to the film, promoting
in the vapor phase than in a liquid state (Guynot et al., 2003; Matan saturation of the system and thus allowing the presence of free
et al., 2006; Tullio et al., 2006). Tunc et al. (2007) observed inhibition molecules. Valencia-Chamorro et al. (2008) established that diffusion
of Penicillium notatum with single and binary combination of aroma effectiveness of an antimicrobial incorporated to an edible film
compounds of carvacrol and cinnamaldehyde. The effect of thyme depends on the polarity of the molecule, its chemical structure, and
EO vapor phase (with a similar concentration of thymol as that the formation of cross-links between molecules, and this would
found in Mexican oregano EO) strongly suppressed the sporulation apply also to volatile compounds. Cagri et al. (2001) reported that
of A. niger during 60 days of exposure at concentrations of 1.00% edible films made with proteins, presented retention of cyclic mole-
(Segvic-Klaric et al., 2006). Similar results were observed in the cules (such as thymol, eugenol, and carvacrol) while Ponce et al.
decrease of growth rate and an increase of lag phase for A. flavus (2008) observed that chemical interactions between the amino
(Bluma et al., 2009). Tzortzakis (2009) studied the inhibition of groups present in casein edible films and carboxyl groups could
 R. Avila-Sosa et al. / International Journal of Food Microbiology 153 (2012) 66–72 71

block the active antimicrobial sites. In our case, chitosan and starch Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in
foods a review. International Journal of Food Microbiology 94, 223–253.
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pounds less effectively and therefore allowed more molecules into properties of low pH whey protein-based edible films containing p-aminobenzoic
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Cagri, A., Ustunol, Z., Ryser, E.T., 2004. Antimicrobial edible films and coatings. Journal
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We acknowledge financial support from the National Council for Holley, R.A., Patel, D., 2005. Improvement in shelf-life and safety of perishable foods by
Science and Technology of Mexico (CONACyT) for the project Combi- plant essential oils and smoke antimicrobials. Food Microbiology 22, 273–292.
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nismos Relacionados con Alimentos. Author Avila-Sosa gratefully
Inouye, S., Tsuruoka, T., Watanabe, M., Takco, K., Akao, M., Nishiyama, Y., Yamaguchi,
acknowledges financial support for his PhD studies from CONACyT H., 2000. Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus
and Universidad de las Américas Puebla. by vapour contact. Mycoses 43, 17–23.
Inouye, S., Takizawa, T., Yamaguchi, H., 2001. Antibacterial activity of essential oils and
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